The gut-associated lymphoid tissue (GALT) is a primary anatomical site for HIV-1 replication, particularly during the early stages of HIV infection, leading to extensive depletion of CD4+ T cells. Accordingly, the principal gut-homing integrin, alpha4beta7 (a4b7), has been identified as an additional cellular receptor for HIV-1 and is emerging as a critical molecule in the pathogenesis of HIV-1 disease. Our previous work demonstrated that IL-7 is a potent inducer of a4b7 expression and activation in both CD4+ and CD8+ T cells. The role of a4b7 in HIV-1 infection is further corroborated by in vivo studies in macaques that documented a protective role of anti-a4b7 antibodies against challenge with simian immunodeficiency virus (SIV). Intravenous administration of a primatized anti-a4b7 monoclonal antibody (mAb), ACT-1, was found to prevent or delay SIV infection in macaques challenged by repeated low-dose vaginal inoculation. Furthermore, administration of anti-a4b7 mAb in the early post-acute phase of infection was recently found to induce persistent control of SIV replication and preservation of the gut lymphoid tissue after withdrawal of antiretroviral therapy (ART). Since HIV-1 has the capacity to incorporate a range of host-cell proteins into its external envelope, which may affect its cellular tropism and infectivity, we investigated the ability of HIV-1 and SIV to incorporate intern a4b7. We found that a4b7 was one of the host proteins most efficiently incorporated by HIV-1, even more efficiently than ICAM-1, which has been extensively characterized in previous studies. As a first step toward elucidating the mechanism responsible for a4b7 incorporation into HIV-1 virions, we investigated the viral components required for such incorporation. Using viral pseudoparticles generated by expression of either the HIV-1 core protein alone (Gag) or Gag plus the envelope glycoprotein (Env) in the presence or absence of a4b7, we found that the presence of Gag alone was sufficient for the viral pseudoparticles to incorporate a4b7, while the addition of Env did not significantly affect the level of incorporation. To evaluate the breadth of incorporation of a4b7 among different clinical and laboratory-adapted HIV-1 or SIV strains, we analyzed a panel of HIV-1 and SIV isolates grown exclusively in PHA-activated primary human PBMC. Incorporation of a4b7 was detected in all the HIV-1 isolates tested, including both clinical and laboratory-adapted isolates, irrespective of their coreceptor-usage phenotype and genetic subtype, as well as in two different SIV strains; in all the viral isolates tested, a4b7 was incorporated more efficiently than ICAM-1/CD54. To determine the functionality of virion-incorporated a4b7, we tested the ability of a4b7+ HIV-1 virions to bind to the natural integrin ligand, MAdCAM-1, in the presence or absence of various modulators of integrin activation such as the cation Mn++. Binding to MAdCAM-1 was detected even in the absence of activating stimuli, indicating that at least a fraction of the incorporated integrin is present in a functionally active state, but treatment with 1 mM MnCl2, which induces a4b7 to adopt a high-affinity ligand-binding state, dramatically increased capture by MAdCAM-armed beads, indicating that the integrin incorporated by HIV-1 is fully functional and responds to physiological stimulations. To investigate if incorporated a4b7 facilitates virion capture and transfer to susceptible target cells, we transfected MAdCAM-1 into MAdCAM-negative cells and tested their ability to mediate trans-infection of susceptible target cells with either a4b7+ or a4b7- virions. HIV-1 infection was detected only when cells expressing MAdCAM-1 were incubated with a4b7+ virus, while all the other conditions did not yield detectable virus transfer to susceptible cells. Furthermore, we demonstrated that incorporated a4b7 can directly promote infection of susceptible MAdCAM-expressing target cells, such as a unique subset of DC in mesenteric lymph nodes, which were recently shown to express MAdCAM-1 in rhesus macaques. To evaluate the physiological relevance of a4b7 incorporation into HIV-1 virions, we investigated whether and to what extent this phenomenon occurs in vivo in HIV-infected patients. Virion incorporation of a4b7 was detected in all patient sera tested, with significantly higher levels in sera taken from patients during the early stage of HIV-1 infection compared with sera from patients during the chronic stage of infection; in contrast, incorporation of LFA-1 was consistently lower in both patient groups. The higher levels of a4b7 incorporation during the early stages of HIV-1 infection are consistent with the high levels of viral replication in intestinal CD4+ T cells, which express high levels of a4b7, during this critical phase of infection. Moreover, we analyzed longitudinal serum samples obtained at weekly intervals from rhesus macaques experimentally infected with SIVmac239 throughout the course of acute primary infection. We found that all the macaque plasma samples contained virion-incorporated a4b7, although incorporation was highest during the acute phase of SIV infection, corroborating the results obtained in infected patients. Finally, to investigate whether virions bearing incorporated a4b7 might specifically home into the intestinal compartment in vivo, where MAdCAM-1 is expressed at high levels, we performed in vivo homing experiments in mice, as murine MAdCAM-1 efficiently binds to human a4b7. HIV-1 virions with incorporated a4b7 were efficiently captured along the lumen of high endothelial venules in intestinal Peyers patches at 45 min post-injection, whereas mice injected with a4b7-negative virus did not show any detectable virus homing to the gut tissue. Virus uptake was specifically inhibited by an anti-a4b7 antibody (ACT-1). These results suggested that a4b7 incorporation may be a critical virulence factor that promotes and sustains HIV-1 infection of the gut compartment, thereby promoting virus spread and pathogenesis, particularly during the early phases of HIV-1 infection.